Genetic instability in cancers frequently occurs at the whole chromosome level, referred to as chromosome instability (CIN). The mitotic checkpoint delays mitosis at metaphase until the attachment of microtubules to the kinetochores is complete, allowing for equal segregation of chromosomes to the two daughter cells. We have recently shown in both primary murine embryonic fibroblasts and human tumor cells that loss of one allele of MAD2, a mitotic checkpoint gene, is sufficient to cause chromosome instability. We hypothesize that disruption of this checkpoint with its attendant aneuploidy will result in deregulated cell growth and may facilitate tumor formation. Therefore, the objective of this proposal is to further elucidate the role that MAD2 plays in the maintenance of genomic stability, tumor initiation and/or progression, and responses to mitotic checkpoint challenges by the use of various genetic approaches. Our specific aims are: (l) To determine the role of haploinsufficiency at the Mad2 locus on cell growth and tumorigenesis in mice. Mad2 +/- mice are completely viable despite the high degree of aneuploidy in MEF's. The effects o f this haploinsufficient phenotype will be studied with respect to spontaneous tumor formation, alterations in cell viability and transformation, and response to spindle disruption and chemical carcinogenesis. (2) To determine if aneuploidy alone is sufficient to alter tumor aggressiveness and whether it can facilitate transformation in human cells. We have used somatic cell gene targeting techniques to inactivate one MAD2 allele and induce aneuploidy in an otherwise chromosomally stable human tumor cell line. The metastatic behavior of these cells and their sensitivity to clinically relevant spindle inhibitors will be studied in a nude mouse model. Similarly, a heterozygous MAD2 deletion will be generated in non-transformed fibroblasts and epithelial cells, and the effect of aneuploidy on cell growth, viability, and transformation will be investigated. (3) To identify the consequences of complete loss of mitotic checkpoint control in mice. Given the embryonic lethality of the Mad2-/- mice, this question will be addressed using the Cre- lox conditional gene targeting approach with subsequent tissues specific deletion of Mad2 in mammary epithelium and B cell lineages. The effects on development, chromosome stability, and tumorigenesis of tissue specific Mad2 null mutations will be studied.